| DCL(Dicer-like) proteins are the members of the conservative dsRNA-specific RNase III family. They are key components in small RNA biogenesis and RNA interference-related processes, and play a pivot role in anti-pathogens defense. These information is mainly from Arabidopsis and rice. In tabacoo, however, DCLs remain uncharacterized. Tobacco is an economically important crop, as well as a model plant species to study plant-pathogen interactions. In this study, we performed functional and expression analyses of the tobacco DCLs, and obtained results mainly as follows:(1) Transgenic tobacco plants carrying RNAi constructs for the four tobacco DCL genes were generated. To this end, RNAi constructs harboring a harpin of DCL gene fragments and suiting for tobacco transformation were made. Employing tissue culture procedure, transgenic tobacco plants carrying RNAi constructs for NtDCL genes (RNAi-NtDCL) were generated,67,33,27and31plants for RNAi-NtDCL1through RNAi-NtDCL4in turn. Detection by PCR for a NPTII fragment in T-DNA and the intron sequence in the harpin of the RNAi constructs revealed that the positive rate of gene transformation is around55%. Analysis of quantative Real-time PCR (qRT-PCR) for DCL gene expression demonstrated that expression of NtDCLs was repressed to a various extent in minimum of only1/10in comparisom with the non-RNAi controls. Investigation of phenotype of the RNAi-NtDCL plants showed that RNAi of NtDCL1, but not other DCLs, affected plant growth and development, resulting in stunted plants, abnormal leaves (slender only-main-vein leaf, curly leaf etc.) and underdeveloped roots. However, silencing of NtDCL2, NtDCL3and NtDCL4in N. tabacum have no obvious developmental defects under normal growth conditions.(2) Expression of DCL genes in avariety of plant tissues was analyzed by qRT-PCR. DCLs are ubiquitously but not evenly expressed in different tissues. The same DCL gene was expressed differently in the different tissues, highly in flower tissues, while lowly in the vegetative organs such as stem, leaf mesophyll, and vein. DCL genes expressed to a different level in same tissues. In the vegetative organs of N. benthamiana, Expression level of DCL2was significantly higher than the other DCLs, followed by DCLI and DCL4, DCL3the lowest. In addition, expression of DCLs differed in the same tissues of the two Nicotiana species. In the stems, veins and other vascular tissues, expression of all the DCL genes was higher in N. tabaccum than in N. benthamiana, while it is not obvious in the leaves and flowers.(3) Functions of DCLs in regulation of plant hypersensitive response (HR) and disease resistance were analyzed employing transient RNAi assays. Compared with non-RNAi control leaves, in RNAi leaves, areas infiltrated with agrobacterium suspension expressing Cf-4/Avr4were deeper green, areas inoculated with Pseudomonas syringae pv. tabaci (Pst) developed wild-fire symptom more quickly and into more severe symptoms, however, areas inoculated with Sclerotinia sclerotiorum (Ss) displayed less severe symptoms. However, there is no obvious difference between the control and RNAi-DCL4leaves, These data revealed that DCL1to DCL3, but not DCL4probably, negatively regulate Cf-4/Avr4-dependent HR and resistance to Ss, while positively regulate resistance to Pst in tobacco, and thus act as important regulators of plant disease resistance. |
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